At the global level, the World Health Organization (WHO) estimates there are ~14 million cases of serious pneumococcal disease and more than 1.6 million people die each year from invasive pneumococcal disease (IPD). Streptococcus pneumoniae has a number of virulence factors that contribute to its ability to cause disease including polysaccharide capsule, pneumolysin and pneumococcal surface proteins A-C. We have recently identified a novel immunoregulatory property of Spn. Our data show exposure of murine effector cells to a soluble fraction generated from mechanically disrupted Spn results in effective inhibition of cytokine production and granule release. The inhibition we observe is a highly novel and unexpected property of pneumococcus. Importantly, this effect is not restricted to a single strain. We have tested an array of clinical and laboratory isolates finding that all could inhibit T effector cell function. In characterizing the inhibitory factor, we found it to be heat labile and protease sensitive. Through a series of fractionation and sequencing approaches, candidate molecules were identified. Two candidates were cloned, engineered to express a His tag, and expressed in E. coli allowing for efficient isolation and further testing. Our data show that one of the candidates could reproduce the T cell inhibitory effect observed with disrupted Spn and in addition, function was found to be independent of the known enzymatic activity of the protein. The ability of Spn to actively regulate function in effector cells presents a new mechanism for immune regulation by this clinically important bacterium. The studies in this application are designed to understand how the differentiation state of the T cell impact susceptibility to the negative regulatory protein. In addition, we propose studies to elucidate the nature of the interaction of the regulatory protein with T cells. Finally, we will determine the susceptibility of human T cells to the negative regulatory capabilities of Spn. These critical questions will be addressed in the following aims. Aim 1 To determine how the differentiation state of a T cell impacts its susceptibility to the immunoregulatory effects of Spn PepN. Aim 2 To determine whether PepN interacts with the extracellular membrane or gains entry into the cell and the potential partners with which PepN interacts. Aim 3 To determine how the presence of Spn components impacts function in human T cells.